Use of functionalised porous membranes or matrices for purification of nucleic acids and corresponding methods

ABSTRACT

Application of a functionalized porous membrane to purify nucleic acids, the binding properties of said membrane with respect to nucleic acids being adjustable by controlling the conditions of an ambient medium, the membrane being functionalized by deprotonated groups, and corresponding methods for purifying nucleic acids.

[0001] The present invention relates to a functionalized membrane or a matrix to purify nucleic acids and to a method for purifying nucleic acids by means of such membranes/matrices.

[0002] Membranes of this species are known from the U.S. Pat. No. 5,438,128. The porous polymer membranes described therein, for instance made of polypropylene or nylon, are functionalized with ion-exchange groups. At low ion concentrations, the functionalized membranes bind to ionized nucleic acids, whereas at high ion concentrations the bound nucleic acids are eluted.

[0003] The known membranes are used in methods or kits to purify nucleic acids. In general the raw material, which may have be pre-purified, containing the nucleic acids, is aspirated or pressed in a low ionic-concentration binding buffer through the porous membrane. When moving through the membrane, the nucleic acids are selectively bound by the ion-exchange groups to the membrane surface. In a next step the membrane is washed optionally with a buffer of higher ionic strength in order to separate unspecifically bound contaminants. The elution of the bound nucleic acids then takes place in a further stage using a buffer of still higher ionic strength.

[0004] The known method incurs the drawback that because of the high salt concentration, the eluate must be purified in almost all cases for instance by ethanol precipitation or dialysis before it may be processed further.

[0005] Accordingly it is the objective of the present invention to create a membrane or a matrix of which the binding properties relating to nucleic acids may be switched and which shall serve to purify nucleic acids in simpler manner, and further to create nucleic-acid purification methods which are matched to said membrane or matrix.

[0006] These goals are attained according to the independent claims 1, 10, 16, 18 and 19.

[0007] The essential feature of the membranes of the invention used to purify nucleic acids is that their surfaces are functionalized with deprotonatable groups. Such membranes are described by ULBRICHT & RIEDEL in BIOMATERIALS 19 (1998), pp 1,229-1,237 in conjunction with protein immobilization. The goal of the invention, namely purifying nucleic acids, was not mentioned or implied at the time.

[0008] The essential feature of the membranes of the invention is that, on account of the functionalization with deprotonatable groups, they exhibit a variably (switchable) adjustable affinity for nucleic acids. It was found that the membrane binding properties can be reproducibly adjusted in particular in pH-dependent manner and/or in relation to ion concentration.

[0009] The membranes may be used for instance to purify nucleic acids of PCR batches, however in general they may used to purify mRNA, total RNA or DNA, in particular plasmid DNA (pDNA) or genomic DNA.

[0010] U.S. Pat. No. 5,693,785 describes using hydroxilated silica beads to purify nucleic acids. However most of the processing protocols disclosed therein require a chaotropic buffer to assure adequate binding of the DNA to the silica beads. In the absence of chaotropic binding buffers binding takes place only when the surface is strongly positively charged. This document neither cites nor makes obvious the possibility of controlling the binding properties by controlling the ambient conditions.

[0011] When using functionalized membranes of the invention, the nucleic acids shall be bound preferably in the absence of chaotropic reagents. The elution of the bound nucleic acids is feasible at low-salt conditions and under some conditions at room temperature, whereby processing is substantially simplified. Still, if so desired, processing in the presence of chaotropic reagents also is possible.

[0012] It was found in practice that for pH values less than or roughly the same as the pK values of their surfaces and/or at raised ionic concentrations, the membranes exhibit good nucleic-acid binding properties. If the pH is shifted toward the alkaline side, and/or the ionic concentration of the ambient medium of the membrane is lowered, then the membrane's affinity for nucleic acids drops and under such circumstances for instance eluting the bound nucleic acids will be feasible.

[0013] As a result the conditions under which the membranes may be used for nucleic-acid purification therefore may be selected in highly diverse manner and be matched to the various applications.

[0014] Another substantial advantage of the invention is that on account of their functionalization, the membranes exhibit hydrophilic surface properties and therefore are well wetting. On account of said good wetting, the volumes of the elution buffers may be kept very small.

[0015] The membrane used in the manner of the invention are functionalized by using groups of sulfonic acid, carboxylic acid and phosphoric acid. Such membranes exhibit especially rigorously switchable surface properties, that is, their affinity for nucleic acids may be well controlled by means of the pH value and/or the ionic concentration of the ambient medium.

[0016] Fundamentally the membrane of the invention with its switchable surface properties already may be constituted by directly configuring the prototonatable groups at or in the membrane surface. The only significant factor in this respect is that the groups shall come into contact with the ambient medium and, depending on conditions in said medium, shall be in the neutral or deprotonated state.

[0017] Preferably however the said groups hall be present at polymer chains, for instance such as polyacrylic acid, of which one end is fixed to the membrane surface and the other end is freely displaceable.

[0018] Membranes of this configuration may assume various boundary conditions. At a pH value less than or the order of the pK value of the membrane surface, i.e. at higher ionic concentrations (for instance >500 mM), the polymer chains rest tightly against the membrane. In this state the nucleic-acid molecules may be adsorbed in especially efficient manner to the membrane surface, presumably pertinent factors being heterogeneous interactions such as van der Waals forces or hydrophobic but also ionic interactions between the nucleic acid and the polymer chain and the polymer chain's functional groups.

[0019] If on the other hand the ambience of the membrane is adjusted to a neutral-to-alkaline pH value and/or to a low ionic concentration, the polymer chains are away from the membrane surface. In this state the bound nucleic acids may be eluted in especially simple manner from the polymer chains, both ionic interactions (the functional groups in the polymer chains are negatively charged and repel the nucleic-acid molecule which is also negatively charged) and possibly as well purely mechanical factors being involved.

[0020] The membranes used in the manner of the invention, in particular are made of plastic. Preferred materials are polypropylene, polyamides, polyesters, polysulfone or PVDF. Conceivably however membranes also may be made of other, for instance inorganic materials such as aluminum oxide, titanium oxide, zirconium oxide or silicon dioxide.

[0021] All solid phases in sheet-form, preferably with a thickness of less than 500 μ, are membranes in the sense of the present invention. Preferably the pore diameter shall be between 0.2 and 10 μ.

[0022] Because the membranes of the invention should be mainly used in high-flow applications, they are designed in a further embodiment mode of the invention for use in micro-titration filter trays, spin columns or reaction containers.

[0023] So far the invention has been discussed with respect to its applicability to membranes. However the fundamental principles also relate to permeable matrices separating nucleic acids.

[0024] Therefore the invention also shall cover the use of such matrices appropriate for purifying nucleic acids which are fitted with a functionalized surface as already discussed above. The above designs of the membrane of the invention also may be applied to such matrices.

[0025] In this respect an especially appropriate matrix would be for instance in the form of a filter. However non-woven webs, felt, fibers or sintered materials may also be used. Moreover correspondingly designed pipet tips or reaction containers (consumables) for instance fitted with micro-structured ducts also might be used.

[0026] The invention furthermore relates to methods for nucleic-acid purification using the above membranes or matrices. In this respect and in the first place, method parameters are stated in three independent claims, whereby nucleic acids may be bound in selective manner. These discussions however also apply to flow-crossed matrices.

[0027] In a first variation, nucleic acid binding is attained by letting a solution of the raw material which contains the nucleic acids permeate a binding buffer of an ionic concentration exceeding 100 mM through the membrane, the pH value preferably being adjusted to be less or roughly that of the pK value of the membrane or matrix surface. During such a flow, the nucleic acids are selectively absorbed by the membrane surface.

[0028] In especially preferred manner, the ionic concentration of the binding buffer is adjusted to be >500 mM. Reproducible quantitative yields are observed at such values.

[0029] In a further variation, the affinity of the membrane/matrix to nucleic acids may be adjusted by means of the pH value. In this variant a binding buffer is selected with a pH such as to be roughly the same as, or less than, or approximately the pK value for instance of the membrane surface. Under these conditions too the nucleic acids are absorbed nearly quantitatively when a solution of the raw material containing the nucleic acids is allowed to permeate the membrane into a binding powder of the cited pH value.

[0030] Lastly the third variation provides placing the raw material into a binding buffer containing a precipitant of a concentration less than the value at which nucleic acids are precipitated. Polyethyleneglycol (PEG) was found being especially appropriate when added to the binding buffer in 6% concentration (PEG 8000). In this case also the nucleic acids bind nearly quantitatively onto the membrane surface.

[0031] In all three variants, adsorption will take place only after the membranes have been correspondingly functionalized. No binding, or no significant binding, takes place as regards unfunctionalized membranes. This behavior is attributed to the cited ambient conditions favoring nucleic-acid binding independently one of another but only in conjunction with the special membrane surface.

[0032] Obviously the individual parameters also may be linked to one another, that is, the membrane designed to bind nucleic acids shall be adjusted to an ambient medium of high ionic concentration and low pH value.

[0033] The binding buffer may be provided separately. However and just as well, it may be combined with the, or one, lysing buffer.

[0034] So far as regards the method of the invention, only the selective binding of the nucleic acids to the membrane was discussed. Desirably however in most applications, and possible following washing, the bound nucleic acids shall also be eluted in simple manner from the membrane. It was again noted in this regard that the above cited independent variants which favor binding the nucleic acids to the membranes can also be used with a reversed sign to elute the bound nucleic acids.

[0035] In the simplest case elution is carried out with water at a neutral pH value. When in the presence of water, the membrane's functional groups are deprotonated in large part and the charges as well as the membrane and also the nucleic acids shall not be shielded. Ionic repulsive motions take place that allow easily detaching the nucleic acids from the membrane. If the membrane or matrix contains the above cited polymer chains where the functional groups are configured, another effect is added, namely the chains point away from the membrane and—in addition to the above described ionic interactions—repel the nucleic acids in purely mechanical manner.

[0036] Obviously other elution buffers are conceivable besides elution with pure water. The expert may readily ascertain which buffer is most appropriate. In general the ionic concentration of the elution buffer shall be as low as possible and the pH value shall be adjusted to be as much above the pK value of the membrane surface as possible. In case of doubt, and if called for, a higher ionic concentration of the elution buffer may be compensated for instance by a more alkaline pH value, and vice versa. Both values can be easily optimized.

[0037] The invention's application of functionalized membranes or the corresponding methods allow especially simple purification of nucleic acids employing conventional stages: binding the nucleic acids to a membrane, washing the membrane and eluting the bound nucleic acids.

[0038] All stages can be carried out in simple manner, under especially gentle conditions and at good yields. In particular the typically required chaotropic reagents of the state of the art and the conventionally employed beads may be eliminated. On account of the membranes and matrices exhibiting good wetting, the elution may be carried out with minimal buffer volumes and for instance at room temperature.

[0039] The invention is particularly applicable illustratively in processing plasmid DNA for purposes of sequencing—a preferred field of application. Clearly other nucleic acids may be purified—where called for following pre-purification from the most diverse raw materials containing such nucleic acids.

[0040] The invention is elucidated below in relation to several Examples which concern especially preferred implementations.

[0041] A: Preparing Functionalized Membranes

EXAMPLE 1

[0042] (a) Modifying Polypropylene Membranes.

[0043] A polypropylene microfiltration membrane (Accurel 2E HF, nominal pore size 0.2 p, membrane thickness 150 μ; Membrana GmbH, Wuppertal [Germany] or test specimen #1333-12A, nominal pore size 0.45 μ; membrane thickness 110 μ, 3M, St. Paul [USA]) having a diameter d=80 mm, while being agitated for 2 h is equilibrated with a 100 mM solution of benzophenone (BP) in acetone. Next the membrane is coated with a 10% aqueous acrylic-acid solution. Then it is illuminated for 15 min with uv radiation (UVA-Spot 2000; Dr Hbnle GmbH, Planegg [Germany]). Finally the modified membrane is extracted with water for 24 h at 60° and dried.

[0044] (b) Modifying Nylon Membranes

[0045] A nylon microfiltration membrane (Schleicher & Schull, nominal pore size 0.45 μ, membrane thickness 127 μ) is modified under the same conditions as in (a) above.

[0046] B: Purifying Nucleic Acids

[0047] The surface-modified membranes prepared in the manner of the above Examples (a) and (b) were clamped into a 96-microfiltration tray during the following purification procedures.

EXAMPLE 2

[0048] The basic material for this test is surface-modified PP (Example 1a) having a nominal pore width of 0.45 μ. Purification is by means of 3,000 rpm centrifuging, room temperature (Eppendorf 581 OR; A-2 DPW rotor) by the bind/wash/elute principle.

[0049] For that purpose 1 μg of pDNA is added each time to the particular 200 μltr of binding buffer 1 (BB1) or 2 (BB2) (BB1: 5 mM tris, pH=7.5; BB2: 4 M NaCI, pH=4.6). Following incubation, washing is carried out twice each time with 200 μltr of 70% EtOH. Elution is carried out with 30 μltr tris (5 mM, pH=7.5). Lastly a second elution with 100 μltr is carried out. Semi-quantitative analysis of 4 μltr eluate is carried out using ethidium bromide gel electrophoresis (omitted).

[0050] Whereas adsorption to the membrane does not take place when using the binding buffer BB1, pDNA when using BB2 may be bound nearly quantitatively to the membrane and may be eluted again in the first elution stage.

EXAMPLE 3

[0051] The test is carried out in the manner described in relation to Example 2, the binding buffers of Table 1 being used. The amount of pDNA used is 500 μg. Semi-quantitative analysis is carried out by means of gel electrophoresis (test results not shown).

[0052] Analysis is shown (in Table 1) and allows a first estimate of the recovery rate (from − to +++). TABLE 1 PP, 0.45μ, hydrophilic, PP, 0.45μ nylon, 0.45μ GH Polypro modified with Binding buffer (S&S), unmodified (Pall Co.) acrylic acid pH = 3.5, 1.5 M (+) − +++ NaCl pH = 7.4, 1.5 M (+) − − NaCl

[0053] As shown by the Table, the DNA may be bound in nearly quantitative manner and then be eluted again only as regards the acrylic-acid modified PP membrane. Table 1 clearly shows the binding's dependency on the pH value of the incubation solution. Binding takes place at a pH value less than the pK value of the membrane modification. No binding takes place at a pH value larger than the pK value.

EXAMPLE 4

[0054] pDNA purification is carried out on the principle of alkaline lysing. For that purpose 1.5 ml of bacterial culture is mixed with the buffers B1-B3 (B1=100 μltr; B2=300 μltr; B3=300 μltr) from the “Perfect Prep Plasmid Midi” kit made by Eppendorf Co. Following centrifuging the clear lysate is mixed each time with 700 μltr of binding buffer (see Table 2). The specimen is processed under the conditions listed in Example 1. The control is in the form of a plasmid preparation, the full purification procedure being carried out with the “Perfect Prep Plasmid Mini” kit of Eppendorf Co., from the identical 1.5 ml of bacterial culture. As a further control, the mixture of clear lysate and the particular binding buffer (Table 2) is centrifuged for 30 min at 12,000 g and at room temperature and the supernatant is removed. Thereupon washing is carried out twice with 200 μltr of 70% ethanol, drying under vacuum and adding 30 μltr tris-HCl, pH=7.5.

[0055] The yield and the purity are determined by photometry and analysis by ethidium bromide gel electrophoresis (Table 2, FIG. 1). This Table lists the concentration of the eluted pDNA in ng/μltr ascertained using a photometer (Eppendorf biophotometer). TABLE 2 Binding buffer: Binding buffer: 6% PEG 6% PEG (mol. wt. = 8,000) (mol. wt. = 8,000) pH = 4.6 pH = 7.4 Nylon, 0.45μ,  8 ng/μltr 15 ng/μltr unmodified (S&S) Nylon, 0.45μ 80 ng/μltr 16 ng/μltr coated with acrylic acid

[0056] The plasmid DNA of the GFP-N1 was analyzed following separation in 0.8% ethidium bromide agarose gel as shown in FIG. 1. The tracks of the gel shown in FIG. 1 were loaded as follows:

[0057] 1) control (without membrane) to show a precipitation mechanism, pH of binding buffer=7.4;

[0058] 2) control (without membrane) to show a precipitation mechanism, pH of binding buffer=4.6;

[0059] 3) unmodified membrane, pH of binding buffer=4.6;

[0060] 4) unmodified membrane, pH of binding buffer=7.4;

[0061] 5) modified membrane, pH of binding buffer=4.6;

[0062] 6) modified membrane, pH of binding buffer=7.4;

[0063] 7) empty

[0064] 8) the control is the plasmid mini preparation (Eppen-dorf Perfect Prep Mini).

[0065] The Gel of FIG. 1 clearly shows that pDNA may only be purified when using the modified nylon membrane. Purification depends on the selected pH value. Thus successful pDNA isolation could be carried out only upon using the binding buffer at pH=4.6, not when it was 7.4.

[0066] Absent a membrane, the precipitation procedure of a mixture of DNA and binding buffer did fail. It may be inferred that the isolation must be attributed to the membrane modification at selected pH conditions, not to a precipitation reaction. 

1. Using a functionalized porous membrane to purify nucleic acids, said membrane's binding properties with respect to nucleic acids being adjustable by controlling the conditions in an ambient medium, characterized in that the membrane is functionalized with deprotonatable groups.
 2. Application as claimed in claim 1, characterized in that the membrane is functionalized with groups of sulfonic acid or carboxylic acid.
 3. Application as claimed in either of claims 1 and 2, characterized in that the groups are bound to polymer chains of which one end is fixed to the membrane's surface and of which the other end is freely displaceable.
 4. Application as claimed in claim 3, characterized in that the fixed polymer chain is a polyacrylic acid.
 5. Application as claimed in one of the above claims, characterized in that the membrane is made of plastic.
 6. Application as claimed in claim 5, characterized in that the membrane consists of polypropylene, polyester, polysulfone, PVDF.
 7. Application as claimed in one of the above claims, characterized in that the diameter of the membrane's pores is between 0.2 and 10 μ.
 8. Application as claimed in one of the above claims, characterized in that the membrane's thickness is less than 500 μ.
 9. Application as claimed in one of the above claims, characterized in that the membrane is designed to be used in microtitration filter trays, spin columns or reaction containers.
 10. Purification of nucleic acids by using a flow-permeable matrix which is fitted with a functionalized surface of which the binding properties relative to nucleic acid are adjustable by means of the conditions of an ambient medium, characterized in that said matrix is functionalized with deprotonatable groups.
 11. Application per claim 10, characterized in that the matrix is a filter.
 12. Application as claimed in either of claims 11 and 12, characterized in that the matrix is in the form of a non-woven, felt, fibers, sintered material, correspondingly designed pipet tips or reaction containers (consumables) with microstructure ducts.
 13. Application as claimed in one of claims 10 through 12, characterized in that the groups are bound to polymer chains of which one end is fixed to the matrix's surface and the other end is freely displaceable.
 14. Application as claimed in claim 13, characterized in that the fixed polymer chain is a polyacrylic acid.
 15. Application as claimed in one of the above claims, characterized in that the matrix is designed for use in micro-titration filter trays, spin columns or reaction containers.
 16. A method for purifying nucleic acids from a raw material containing nucleic acids as claimed in claims 1 through 9 or a matrix as claimed in claims 10 through 15, where a raw-material solution in a binding buffer of high ionic concentration (>100 mM) is allowed to permeate the membrane/matrix, the nucleic acids selectively binding to the surface of the membrane/matrix and optionally and in a further stage again eluting the bound nucleic acids from the membrane/matrix.
 17. Method as claimed in claim 16, characterized in that the ionic concentration of the binding buffer exceeds 500 mM.
 18. Method for purifying a raw material's contained nucleic acids using a membrane as claimed in claims 1 through 9 or using a matrix as claimed in claims 10 through 15, where a solution of the raw material in a binding buffer is allowed to permeate the membrane/matrix of which the pH value is less than or roughly the same as the pK value of the surface of the membrane/matrix, the nucleic acids selectively binding to the surface of the membrane/matrix, and optionally the bound nucleic acids being eluted again from the membrane/matrix in a further stage.
 19. Method for purifying a raw material's contained nucleic acids using a membrane as claimed in claims 1 through 9 or a matrix as claimed in claims 10 through 15, where a solution of the raw material in a binding buffer is allowed to permeate the membrane/matrix, said buffer containing a precipitant in a concentration less than the value at which the nucleic acids will precipitate, and optionally the bound nucleic acids are eluted again from the membrane/matrix in a further stage.
 20. Method as claimed in claim 19, characterized in that the binding buffer contains PEG in a concentration less than 10%.
 21. Method as claimed in one of the above claims, characterized in that an elution buffer, of which the pH value is adjusted to exceed the pK value of the membrane/matrix surface, is allowed to permeate the membrane/matrix in order to elute the bound nucleic acids.
 22. Method as claimed in one of the above claims, characterized in that an elution buffer, of which the ionic concentration has been adjusted to less than 500 mM, is allowed to permeate the membrane/matrix for the purpose of eluting the bound nucleic acids.
 23. Method as claimed in one of the above claims, characterized in that the binding-, washing- and/or elution-buffer is vacuum aspirated through the membrane/matrix.
 24. Method as claimed in one of the above claims, characterized in that the elution of the nucleic acids bound at the membrane/matrix is carried out at room temperature.
 25. Method as claimed in one of the above claims, characterized in that the raw material is subjected to pre-purification in order to remove coarse cell debris and the like.
 26. Method as claimed in the preceding claim, characterized in that the raw material is a clarified cell lysate. 